Simian virus 40 (SV40) can cause tumors when injected into susceptible hosts. We have proposed to isolate new SV40 mutants which are defective in the genes coding for two tumor antigens, t and T. These new mutants will be used to study the role of these putative oncogenic proteins in cell transformation by SV40 and in oncogenesis. Rat cells transformed by new mutants with genotypes t-Tts and ttsT- will be used to determine which transformed cell phenotypes are under the maintenance control of t or T. Immunocompatible rats will be inoculated with rat cells transformed by mutants with genotypes t-T plus and t plus T- to determine whether t or T (or both) is involved in SV40 oncogenesis. Finally, these mutants will be used to study the mechanisms by which these putative proteins may act to cause cell transformation and tumors. We have isolated 159 mutants with deletions in the gene for t (but not T) ranging in size from 3% to 5% of the genome. These have been characterized and we have selected 29 mutants that have the lowest efficiency of transformation of cells to grow in soft agar. We are screening these for temperature-sensitivity. We have constructed several mutants with large deletions in the distal end of the gene for T and are characterizing the genomes of these mutants by fine structure restriction enzyme mapping and DNA sequence analysis. We are testing these mutants to determine if a normal t protein is made and whether T antigen, albeit truncated, is present in infected cells. We have constructed a t-Tts double mutant by joining together the defective regions of two existing mutants, dl884 and tsA58. This mutant does not produce a detectable t protein but does produce a thermolabile T antigen of normal size. We are using this mutant in transformation studies. We are attempting to isolate a ttsT plus mutant by mutagenizing the t-specific region of the genome, are characterizing all of our mutants, and will use them during the second two years of grant period.